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CRTH2 and CD161 define a human IL-25 and
IL-33–responsive type 2 innate lymphoid cell type
Hergen Spits, Jenny Marie Mjosberg, Sara Trifari, Natasha Crellin, Charlotte
P Peters, Cornelis M van Drunen, Berber Piet, Wytske J Fokkens, Tom
Cupedo
To cite this version:
CRTH2 and CD161 define a human 25 and
IL-33−responsive type 2 innate lymphoid cell type
Jenny M Mjösberg1, Sara Trifari4, 6, Natasha K Crellin4, Charlotte P Peters1, Cornelis M van Drunen2, Berber Piet3, Wytske J Fokkens2, Tom Cupedo5 & Hergen Spits1
1Tytgat institute for Liver and Intestinal Research, Departments of 2Otorhinolaryngology
and 3 Experimental Immunology and Pulmonology of the Academic Medical Center,
University of Amsterdam, the Netherlands
4Department of Immunology Genentech, South San Francisco, California, USA 5Department of Hematology, Erasmus University Medical Center, Rotterdam, the
Netherlands
6Present addresses: La Jolla Institute for Allergy and Immunology, La Jolla, California,
SUMMARY
INTRODUCTION
Innate lymphoid cells represent an emerging family of cell types that appear to play crucial roles in tissue remodeling and in innate immunity against pathogenic and non-pathogenic micro organisms1-3. These cells are characterized by a lymphoid morphology and an absence of recombination activating gene (RAG)−dependent receptors. Natural killer (NK) cells and Lymphoid Tissue inducer (LTi) cells are the prototypic members of this family. Whereas NK cells have a crucial innate defensive role against viral infections, in particular against herpes viruses, LTi cells are essential for the formation of lymph nodes during embryonic development. Initially LTi cells were not considered to have a function in the immune response. However, the recent discovery in mouse and humans that LTi cells are able to produce interleukin 17 (IL-17) and IL-22 (refs. 4-6), cytokines that are known to mediate immunity against microbes, has led researchers to reconsider the functions of these cells in immune responses. More recently, cells were discovered that show characteristics of both NK and LTi cells. Like LTi cells these cells are of lymphoid origin, express the IL-7Rα chain (CD127) and the transcription factor RORγt but they also share with NK cells the expression of NKp46 and, in humans, NKp44 and CD56 (refs. 7-10). These NK receptor−expressing cells are mostly found at mucosal sites both in mice and humans and produce IL-22. Innate lymphoid cells (ILCs) dedicated to the production of IL-17 and interferon-γ (IFN-γ), either exclusively or, in combination, have been described both in mice and humans11-13. IL-22-producing ILCs
bacterium Citrobacter rodentium in the gut14 and Klebsiella pneumoniae in the lung15. It seems therefore that the ILC family comprises cells that share a number of characteristics but may have different effector functions mediated by distinct cytokines and dependent on their anatomical localization3.
Recently, another Id2-dependent non−T, non−B cell population which produces type 2 cytokines was discovered in the mouse. These cells have been designated natural helper lymphocytes (NHLs)16, multi-potent progenitor type 2 (MPP2) cells17,
nuocytes18 and innate helper (IH) type 2 cells19. Although differences exist between these
cell types, they have in common the production of TH2 cytokines, most notably IL-5 and
IL-13 in response to the IL-17 family member IL-25 (IL-17E) and the IL-1 family member IL-33. The innate lymphocyte−derived TH2 cytokines mediate eosinophilia and
goblet cell hyperplasia, both of which are critical for anti-helminth reactions. These type 2 ILC populations may be involved in disease since their signature cytokines 5 and IL-13 also play a role in pathophysiology of type 2 immunity−associated diseases such as asthma and allergic diarrhea.
Like LTi cells and RORγt+ ILCs9,20, type 2 ILCs are dependent on the gamma common cytokine receptor (γc) and IL-716,21. Type 2 ILCs depend on Id216 similar to LTi
and IL-17− and IL-22−producing ILCs22,23, but in contrast to those cells type 2 ILCs seem to be independent of RORγt16. The common reliance of NK cells, LTi cells and various ILC populations on Id2 and γc cytokines support the contention that these cell types have a common origin3.
Previously it was shown that cord blood CD34+ progenitor cells cultured in the presence of IL-2, can develop into a non−T, nonB cell type that expresses CD161but lacks CD56 (ref. 24). These cells produced IL-13 but not IFN-γ, and contained an IL-5–producing subset. However, an equivalent cell type was not identified in vivo. More recently, we demonstrated that lineage (T, B and monocytes)–negative (Lin–) CD127+CD117+ cells from tonsil are able to produce IL-13 following stimulation with TLR2 ligand and IL-2 (ref. 13). These IL-13−producing human ILCs expressed RORC transcripts and protein and expressed IL-22. Cloned lines were established from these cells which mostly co-expressed IL-22 and IL-13, although some clones were identified that produced IL-13 and IL-5 but not IL-22 (ref. 13). These latter clones still expressed RORC transcripts, in contrast to mouse type 2 ILCs. We also found that ILCs from the tonsil when stimulated with IL-23 and IL-2 produced IL-22 but not IL-13 whereas when these ILCs were simulated with IL-2 and TLR2 agonists they do produce IL-13 (ref. 13) suggesting that tonsil ILCs are plastic and produce different cytokines dependent on the stimulus given to those cells. It is, however, also possible that tonsil ILCs contain two subsets, one of which produces IL-13 and does not respond to IL-23. Obviously a marker discriminating IL-13-producing from IL-22 –producing ILCs would allow for addressing this issue.
Here we describe a human Lin−CD127+ ILC population that is characterized by the expression of the TH2 marker Chemoattractant receptor−homologous molecule
expressed on TH2 lymphocytes (CRTH2). These ILCs were found in lung, gut and nasal
these human CRTH2+ cells responded to IL-25 and IL-33 by producing IL-13 and IL-5 and we therefore suggest that these cells represent the human equivalent of mouse type 2 ILCs.
RESULTS
The fetal gut contain a lin-CRTH2+CD127+ population
In the mouse type 2 ILCs have been found in the intestine. In search of a human cell population that may represent the equivalent of the type 2 ILCs described in the mouse we performed an extensive analysis of cells in both fetal and adult intestines, focusing on cells that were Lin– and IL-7Rα+. Thus, we stained single cell suspensions derived from intestinal tissues with a cocktail of lineage specific antibodies, CD45 and a collection of antibodies, including the antibody against CRTH2 which is known to be expressed on TH2 cells and myeloid cells associated with type 2 immune responses such as basophils
and mast cells. Unexpectedly, we found the highest proportion of Lin−CRTH2+ lymphoid cells in the fetal gut and therefore decided to perform an extensive analysis of all ILC populations within this organ. Within the Lin– population, we could clearly distinguish cells with high and cells with intermediate expression of CD45 (CD45high versus CD45dim) (Fig. 1a). Analyzing these two populations for CD117 (c-Kit) and CD127 (IL-7Rα) expression we observed that the great majority (>90%) of the CD45dim cells co-expressed CD117 and CD127 and most likely contain LTi4. By contrast, only around
gated on CD127+ cells and analyzed the CD45high Lin– cells within that gate and compared these with CD127+CD45dim cells. Both populations expressed CCR6, CCR4, the lymphoid marker CD7 and CD161, which are also expressed on NK and LTi cells (Fig. 1b). Thus the CD45highCD127+ cells most likely represent an ILC population. The CD45dim cells, like LTi cells in the mesenteric lymph nodes, expressed IL-1βR and contained two populations, one that expressed NKp44 and the other lacking this antigen (Fig. 1b). By contrast, the fetal gut CD45high cells all lacked NKp44 and expressed low amounts of IL-1βR (Fig. 1b). The great majority of these CD45highCD127+ cells were clearly positive for the type 2 marker CRTH2 (Fig. 1c). To assess whether these CRTH2+ cells were not mast cells or basophils, that also express CRTH2+, we analyzed the CRTH2+ ILCs for the presence of the IL-3 receptor (CD123) and the FcεRI which are normally highly expressed on those cell types. The CRTH2+ lymphoid cells did not bind IL-3R− nor FcεRI−specific antibodies (Fig. 1c), indicating that these cells are distinct from both mast cells and basophils.
Fetal LTi cells and postnatal IL-17− and IL-22−producing ILCs express and depend on the transcription factor RORγt, whereas type 2 ILCs in the mouse have been shown to be negative for this transcription factor. Therefore we analyzed the expression of RORγt on the various ILC populations. Since this antibody has a high non-specific binding activity we used the binding to NK cells, which do not express RORγt as assessed by real-time PCR analysis, as background. CD45dimCD127+ ILCs were RORγt positive whereas NK cells (CD45+CD127−CD56+ cells) were negative for this factor (Fig. 1d). The CRTH2+
expressed less RORC transcripts than CD45dimCD127+ ILCs, but still more than NK cells (Fig. 1d). Thus, a CRTH2+ ILC population, distinct from RORγt+ ILC, mast cells and
basophils can be found in fetal gut.
Fetal gut CRTH2+ ILCs respond to IL-25 and IL-33
The signature cytokines of human and mouse RORγt+ ILCs are IL-17 and IL-22 (reviewed in 3) whereas mouse type 2 ILCs secrete IL-13 (refs. 16,18,19). We therefore analyzed the expression of transcripts for these cytokines and IL-5 in the various ILC populations isolated from the fetal gut. Whereas CD45dimCD127+NKp44+ expressed
IL-22 and some IL-17 transcripts they did not express IL-13 (Fig. 2). By contrast, CRTH2+ ILCs clearly expressed IL-13 but no IL-17 or IL-22 transcripts. Neither the CRTH2+ population nor the other ILC populations in the gut expressed IL-5 ex vivo (data not shown). Since CRTH2+ cells express CD25 and IL-13, we stimulated freshly isolated fetal gut CRTH2+ cells, with 2, and the combination 2 plus 25 or 2 plus
IL-33. IL-2 in combination with IL-25 or IL-33 clearly stimulated production of IL-13 protein (Fig. 3).
cell, T cell or NK cell lineages (Fig. 4a). In vitro expanded CRTH2+ ILCs expressed intracellular IL-13 but no IL-17 following activation with the polyclonal activator phorbol 12-myristate 13-acetate (PMA) and ionomycin whereas a fraction of the 13−producing cells also produced 22 (Fig. 4b). As previously reported for IL-22−producing NKp44+ ILCs, CRTH2+ ILC cell lines expressed aryl hydrocarbon receptor (AHR) (Fig. 4c). However, CRTH2+ ILCs did not respond to IL-23 (Fig. 4d), in
contrast to NKp44+ ILCs isolated from inflamed tonsils (Fig. 4e), to elicit production of IL-22 or expression of IL17 (data not shown), consistent with the lack of the IL23R (Fig.
4f).
CRTH2+ ILC cell lines clearly expressed ST2 (also known as IL1RL1, subunit of IL33R),
IL17RB (subunit of IL25R) and IL17RA (common subunit of IL25R and IL17R)
transcripts (Fig. 4f). Consistent with the expression of ST2, the majority of the cell lines clearly responded to IL-2 in combination with IL-33 and IL-25 plus IL-33 (Fig. 4g) whereas response to IL-25 was less pronounced and not seen in all cell lines (Fig. 4g and
Supplementary Fig. 1). The responses of individual cell lines are shown in
Supplementary Fig 1.
The CRTH2+ ILC cell lines never showed any IL-17 expression (data not shown) but in contrast to the ex vivo isolated cells, the cell lines did express IL-5 transcripts (data not shown). Thus, we have identified a stable Lin– CRTH2+ CD127+ population that expresses IL-13 but not IL-17 or IL-22 transcripts ex vivo and respond in vitro to IL-25 and IL-33 by producing IL-13.
To gain more insights into the possible functions of CRTH2+ ILCs, we performed an extensive analysis of the presence of Lin−CRTH2+CD127+ in a variety of tissues. These cells were located in mucosal tissues at different ontogenic stages as they were found in both fetal and adult gut and lung (Fig. 5a and Table 1).
Given the production of type 2 cytokines and the responsiveness to IL-25 and IL-33, we analyzed the presence of CRTH2+ cells in chronically inflamed airway tissues, specifically nasal polyps of patients with chronic rhinosinusitis (CRS). This disease is characterized by the presence of very high local titers of IgE and high numbers of eosinophils that may be driven by the eosinophil growth factor IL-5 and further supported by IL-13. IL-5 and IL-13 transcripts are indeed elevated in these patients compared to chronic rhinosinusitis patients without nasal polyps27. Indeed, nose polyps contained relatively high proportions of CRTH2+CD127+CD161+ ILCs when compared to non-inflamed nose tissue from healthy, non-allergic donors (Fig. 5b and Table 1). These data support the hypothesis that CRTH2+ cells are type 2 ILCs and that they can contribute to type 2 mediated disease. In contrast to CRTH2+ ILCs in peripheral blood, which express low amounts of RORγt, the nose polyp−residing CRTH2+ cells did not seem to express
any RORγt protein (Fig. 5d), an observation that associates these cells to the mouse natural helper cells which were reported to be completely negative for RORγt16.
CRTH2+CCR6+ ILCs are present in peripheral blood
That the CRTH2+ ILCs are present in many tissues may mean that they differentiate in
presence of Lin−CRTH2+ and other ILCs. For this analysis we depleted peripheral blood mononuclear cells (PBMCs) of the majority of T (with anti-CD3), B (anti-CD19) cells and monocytes (anti-CD14). When gating on Lin– cells, a population of CD127+ cells, that is distinct from CD56−expressing NK cells were clearly present (Fig. 6a). Within the Lin–CD127+ population, we could distinguish subsets of CD117+ and CD117– cells. To investigate how the peripheral Lin−CRTH2+ cells may be related to the cells we found in other human tissues, we analyzed the expression of comprehensive panel of markers. CD127+CD117+ cells abundantly expressed the lymphoid markers CD7, CD161, CD25 and CD62L (Fig. 6b). They were negative for NKp44, thus suggesting they are different from the previously described human IL-22−producing NKp44+ cells, and expressed low amounts of HLA-DR. The CD127+CD117− population was very similar to the CD127+CD117+ population with respect to these markers, although some, such as CD25 and CD161, clearly was present in a bimodal distribution (Fig. 6b), suggesting that this population is heterogeneous. Neither CD127+CD117+ nor CD127+CD117− cells expressed CD34 (results not shown), indicating that these cells populations do not contain immature hematopoietic progenitors. Both the CD127+CD117+ and the
CD127+CD117− populations expressed CCR6 and CRTH2 (Fig. 6c), two receptors also found on fetal gut type 2 ILCs (Fig. 1b). By contrast, CRTH2 was not present on neither CD56high nor CD56+ NK cells (Supplementary Fig. 2). The expression of RORγt protein on CRTH2+ cells was lower than that expressed in TH17 cells but by comparison to NK
cells, which lack RORγt, the CRTH2+ cells expressed some RORγt (Fig. 6d), which is in
indicated that human peripheral blood contains a cell population that shares the main phenotypic features of tissue resident type 2 ILCs.
Blood CRTH2+ ILCs respond to IL-25 and IL-33
Lin−CD127+CD117+ circulating cells stimulated with PMA plus ionomycin produced a wide range of cytokines, including IL-2, IL-13, tumor necrosis factor (TNF) and low amounts of IL-22 and IL-17 (Supplementary Fig. 3), suggesting that these cells are functionally heterogeneous. Indeed, when we stimulated freshly isolated cells with IL-2 only, or a combination of IL-2 plus IL-25 or IL-2 plus IL-33, IL-13 was induced only in the CRTH2+ but not in the CRTH2− subset (Fig. 7). To determine the stability of these cells, CRTH2+ cell lines were generated from these cells. All expanded cells expressed CRTH2 and CD127 (Fig. 8a) and weakly expressed RORC transcripts (Fig. 8b) similar to ex vivo isolated CRTH2+ ILCs, confirming the stability of this phenotype. The cultured cells expressed large amounts of IL-13 when stimulated with the polyclonal stimulus ionomycin plus PMA but did not express IL-17 (Fig. 8c). Furthermore these cell lines expressed ST2, IL17RB and IL17RA (Fig. 8d). CD117- cells expressed more
associated with IL-22 production (Fig. 8f). However, stimulation with IL-23 or IL-1β did not further enhance IL-22 secretion (Fig. 8g), in contrast to what was seen for tonsil NKp44+ cells (Fig. 4e). Taken together our data indicate that part of the CRTH2+ cells from peripheral blood has the ability to produce IL-22, in contrast to the freshly isolated CRTH2+ ILCs from fetal gut.
DISCUSSION
Here we describe CRTH2+ innate lymphoid cells that are distinct from basophils and mast cells.. Lin−CRTH2+ ILCs are present in intestinal tissue, during the fetal stage, and persist in adults. Ex vivo isolated fetal gut CRTH2+ ILCs expressed IL-13 but not IL-17 nor IL-22. In vitro, these cells responded to IL-25 and IL-33 by producing the type 2 cytokine IL-13. Our data suggest therefore that the CRTH2+ ILCs described here are similar to a non−B, non−T, γc-dependent IL-25−responsive cell type that was identified previously21 and more recently rediscovered by several other groups, who named these cells nuocytes18, natural helper lymphocytes (NHL)16 and innate helper 2 cells (ih2)19, respectively.
inflammation. In this respect our observation of relatively high proportions of type 2 ILCs in nasal polyps of rhinosinusitis patients is relevant as this disease is characterized by eosinophilia, most likely caused by IL-5. Furthermore, IL-5 and IL-13 transcripts are elevated in these patients compared to chronic rhinosinusitis patients without nasal polyps27.
Since CRTH2+ ILCs are also localized in the gut, it is conceivable that these cells may play a role in chronic gut inflammations with an IL-13−linked etiology, such as ulcerative colitis (UC)29. Interestingly, anti-CD161 treatment of lamina propria (LP) cells from UC patients resulted in a 90% reduction in amounts of IL-5 and IL-13 (ref 30). It was concluded that the CD161+ NKT cells were the main producers, however it may be possible that type 2 ILCs that also express CD161 contribute to IL-13 production in UC tissues. Future work analyzing the function of CRTH2+ cells in other type 2 immunity−mediated inflammatory diseases such as asthma, allergic diarrhea and atopic skin disorders should reveal the possible role of these cells in type 2 immune−mediated inflammatory diseases possibly providing new avenues for therapeutic intervention directed at these cells.
have also been implicated in regeneration of lymphoid tissue after acute viral infections31 and it is possible that type 2 ILCs also have such a function as well.
The presence of CRTH2+ cells in a variety of tissues raised the question where these are derived from circulating mature CRTH2+ ILCs. Addressing this issue we observed that IL-25 and IL-33−responsive CRTH2+ cells with similar phenotypes as those found in the tissues were present in the peripheral blood. However, the peripheral blood type 2 ILCs did not express IL-13 or other cytokine transcripts ex vivo in contrast to what we observed in fetal gut, indicating they are in a non-activated state. The majority of CRTH2+ peripheral blood cells co-expressed CCR6 which may be instrumental in homing of these cells to the tissues. It is possible that type 2 ILC homing in different tissues get activated in situ and therefore adopt slightly different features such as expression of IL-13 and perhaps certain cell surface receptors which might explain why type 2 ILCs found in different tissues in the mouse are not identical to each other32. Further supporting this idea, we observed a clear-cut IL-13 producing profile of gut tissue type 2 ILCs whereas a minority of the circulating CRTH2+ ILCs also produced some IL-22, which was not regulated by IL-1β or IL-23, further supporting that the circulating ILC population is distinct from the previously described IL-22 producing ILCs but have more functional plasticity than tissue resident type 2 ILCs.
Compared to NK cells that do not express transcripts of RORC nor RORγt protein, CRTH2+ type 2 ILCs express some RORγt as determined by flow cytometry and analysis of RORC transcripts. This observation was unexpected since mouse type 2 ILCs were reported to be negative for RORC transcripts16. In contrast to fetal LTi cells and post natal IL-17 or IL-22−producing ILCs, type 2 ILCs are also independent of RORγt for their development in the mouse16. The expression of RORγt in human CRTH2+ cells does not necessarily mean that these cells depend on this transcription factor. It is possible that type 2 ILCs and IL-17, IL-22−producing ILCs have a common RORγt+ precursor; whereas differentiation of the common precursor to IL-17, IL-22−producing ILCs requires RORγt, this might not be the case for type 2 ILCs. An analogous situation exists in the T cell system. RORγt is expressed on all double positive thymocytes33. Although RORγt is required for optimal survival of DP thymocytes, mature TH cells do develop in
RORγt deficient mice except for TH17 cells34. Published evidence exists to suggest that
RORγt expression is not always stable35; using RORγt reporter mice these researchers observed that downregulation of RORγt was associated with a functional shift from IL-22 to IFN-γ production. Perhaps an analogous situation exists for CRTH2+ ILCs in that they
may develop from RORγγt+ ILCs whereby downregulation of RORγt and IL-22 production parallels upregulation of CRTH2 and 13. The question whether or not IL-17, IL-22−producing ILCs and type 2 ILCs derive from a common RORγt+ precursor should be addressed in mouse models with cell fate mapping experiments.
present in inflamed nasal polyps of chronic rhinosinusitis, a type 2 immune mediated inflammatory disease27. These cells may therefore be the human equivalent of cells recently found in the mouse which were termed natural helper cells, nuocytes or innate helper cell type 2.
Acknowledgements
We thank B. Hooibrink (AMC) and J. Cupp (Genentech) and their teams for their help with flow cytomery and Luminex assays. We also thank W. Ouyang and C. Kaplan (Genentech) for stimulating discussions. A. te Velde and C. Ponsioen are acknowledged for their help with intestinal tissues. The staff of the Bloemenhove clinic in Heemstede, the Netherlands is acknowledged for providing fetal tissues and we thank K. Weijer, A. Voordouw, N. Legrand and B. Olivier for their help with processing various tissues.
Author contributions
Figure legends
Figure 1 Lineage negative lymphocytes in the fetal gut contain a CRTH2+CD127+
ILC population.
(a) Flow cytometry analysis of two distinct ILC populations in the fetal gut. Lin (CD1a, CD3, CD11c, CD14, CD19, CD34, CD123, TCRαβ, TCRγδ, BDCA2, FcεR1) negative cells were gated as 1) CD45dim or 2) CD45high and analyzed for expression of CD127 and CD117. (b) Flow cytometry characterization of Lin-CD127+CD45dim (grey line) and Lin -CD127+CD45high (black line) as compared to isotype control (shaded light grey). (c) Flow cytometry analysis of CRTH2, CD117, CD123 (IL-3R) and FcεR1 expression on CD45highLin-CD127+ cells (black line) and peripheral blood monocytes or basophils in (light grey thin line). Isotypes are shown in shaded light grey. (d) Flow cytometry analysis of RORγt protein expression in CD56dim peripheral blood NK cells (dashed grey line), fetal gut CD45highLin-CD127+CRTH2+ ILCs (black solid line) and CD45dimLin -CD127+NKp44+ ILCs (grey solid line). Data are representative of at least 5 fetal donors. Right panel in (d) shows RORC mRNA expression in fetal gut CRTH2+ ILC (gated
CD45highLin-CD127+) as compared to fetal gut NKp44+ ILCs, NKp44- ILCs (gated CD45dimLin-CD127+CD117+), conventional NK cells (cNK, CD45highCD127-CD56+), tonsil NKp44+ ILCs and fetal mesenteric lymph node (MLN) NKp44- ILCs. Data are shown as median and range (n=2-5).
Fetal gut CRTH2+ ILCs (gated CD45highLin-CD127+) were purified by flow cytometric sorting and analyzed with real-time PCR for expression of IL13, IL22, IL17, IFNG and
TNF transcripts as compared to the expression in fetal gut NKp44+ ILCs, NKp44- ILCs (gated CD45dimLin-CD127+CD117+), conventional NK (cNK, CD45highLin-CD127 -CD56+) cells, tonsil NKp44+ ILCs and fetal mesenteric lymph node (MLN) NKp44- ILCs. Lineage cocktail included CD1a, CD3, CD4, CD11c, CD14, CD19, CD34, CD123, TCRαβ, TCRγδ, BDCA2 and FcεR1. Data are shown as median and range and were obtained from 2-5 donors.
Figure 3 CRTH2+ fetal gut ILCs respond to 25 and 33 with production of
IL-13 protein in vitro.
(a) In experiment 1, fetal gut CD45highLin-CD127+CRTH2+ ILCs were sorted by flow cytometry and cultured for 4 days with 2 (10 U/mL) or a combination of 2 and IL-25 (10 U/mL and 50 ng/mL, respectively). (b) In experiment 2, CD45highLin -CD127+CRTH2+ ILCs were sorted also on basis of CD117 and stimulated as in a. (c) Stimulation of fetal gut CD117+CRTH2+ and CD117-CRTH2+ ILCs with IL-2 or a
combination of IL-2 and IL-33 (10 U/mL and 50 ng/mL, respectively). Supernatants were analyzed for IL-13 protein by ELISA. Concentrations were normalized for 2000 cells/200 µL in a 96-plate well. Lineage cocktail for flow cytometry sorting included CD1a, CD3, CD4, CD11c, CD14, CD19, CD34, CD123, TCRαβ, TCRγδ, BDCA2 and FcεR1.
(a) Flow cytometric analysis of expanded fetal gut CD45highLin- (CD1a, CD3, CD4, CD11c, CD14, CD19, CD34, CD123, TCRαβ, TCRγδ, BDCA2, FcεR1) CD127+CD117+CRTH2+ ILC cell lines (black lines, isotype controls in shaded light grey). (b) Flow cytometry analysis of PMA−Ionomycin stimulated fetal gut CRTH2+ ILC cell lines (black lines, unstimulated cells in shaded light grey) and CD56+ conventional NK cell line (grey lines) stained for intracellular IL-13, IL-22 and IL-17. Far right plot shows PMA−ionomycin stimulated fetal gut CD127+CD117+CRTH2+ ILC cell line. Data are representative of 2-3 fetal donors. (c) Expression of AHR mRNA in fetal gut CRTH2+ ILC, blood CD56dim conventional NK cells (cNK) and tonsil NKp44+ ILC cell lines (data shown as median and range, n=3-4). (d) IL-22 secretion from fetal gut CRTH2+ ILC or (e) tonsil NKp44+ ILC cell lines following stimulation with IL-1β (50 ng/mL), IL-23 (50 ng/mL) and combinations of these (data shown as mean and standard error, n=3-4). (f) Expression of ST2, IL17RB, IL17RA and IL23R mRNA in fetal gut CRTH2+ ILC, blood CD56dim conventional NK cell (cNK) and tonsil NKp44+ ILC cell lines (data shown as median and range, n=3-5). (g) IL-13 response of fetal gut CRTH2+ ILCs to IL-25, IL-33 and combinations of these (n=3).
Figure 5 CRTH2+ ILCs are distributed in several fetal and adult tissues and are
enriched in nose polyps of chronic rhinosinusitis (CRS) patients.
(a) Mononuclear cells were isolated from fetal gut, fetal lung, adult gut and adult lung, stained for ILCs and analyzed by flow cytometry. ILCs were gated as CD45+Lin-(CD1a,
are representative of 2-5 donors. (b) Shows CRTH2+ ILCs in healthy control (HC) nasal tissue (left dot plot) and chronic rhinosinusitis (CRS) nasal polyps (right dot plot). Differences in CRTH2+ ILC frequencies were calculated using Mann-Whitney two-tailed test. ILCs were defined as CD45+Lin- (CD1a, CD3, CD11c, CD14, CD19, CD34, CD123, TCRαβ, TCRγδ, BDCA2, FcεR1) CD127+ and analyzed for expression of (c) CD161 and (d) RORγt. The figures in c-d are representative of 4 CRS patients.
Figure 6 CRTH2+CD127+CCR6+ innate lymphoid cells are present in peripheral
blood.
(a) Flow cytometric analysis of peripheral blood cells that were pre-depleted from the majority of T, B cells and monocytes; gating shows the presence of a Lin−CD127+CD117+ and a Lin−CD127+CD117− populations (right plot); (b) flow cytometric analysis of the indicated markers in the Lin−CD127+CD117+ and a Lin−CD127+CD117− populations, gated as in (a); Shaded grey indicate isotype control Ab; (c) Flow cytometric analysis of CCR6 versus CRTH2 expression in Lin−CD127+CD117+ and a Lin−CD127+CD117− cells, gated as in a; (d) Expression of
RORγt protein in lin-CD127+CRTH2+ ILCs (black line), conventional CD56+ NK cells (dashed grey line) and TH17 (CD3+CD4+CCR4+CCR6+) cells (solid grey line). All plots
are representative of at least 3 independent donors.
Figure 7 CRTH2+ peripheral blood ILCs respond to IL-25 and IL-33 with
PBMC were depleted from T (CD3), B (CD19), NK (CD16) cells and monocytes (CD14) using magnetic beads. Lin-CD127+CRTH2+ and Lin-CD127+CRTH2- cells were sorted and cultured for 4 days with (a) IL-2 (10 U/mL) or a combination of IL-2+IL-25 (10 U/mL+50 ng/mL) and (b) IL-2 (10 U/mL) or a combination of IL-2+IL-33 (10 U/mL+50 ng/mL). Supernatants were analyzed for IL-13 protein by ELISA. Concentrations were normalized for 2000 cells/200 µL. Data shown as mean and standard error (n=2-6). Lineage cocktail used for sorting included CD1a, CD3, CD4, CD11c, CD14, CD19, CD34, CD56, CD94, CD123, TCRαβ, TCRγδ, BDCA2 and FcεR1.
Figure 8 Stable cell lines can be generated from CRTH2+ peripheral blood ILCs.
Table I. Tissue distribution of CD45+Lin-CD127+CRTH2+ ILCs Tissue CD45+Lin-CD127+CRTH2+ % of CD45+ (range) Fetal gut (n=5) 0.5-2.3 Fetal lung ( n =2) 0.2-0.3 Adult ileum ( n =5) 0.01-0.1 Adult lung ( n =3) 0.02-0.08 Adult blood ( n =3) 0.01-0.03
Supplementary table I. Sequences of real-time PCR primers designed in house.
* Kindly provided by M. Vondenhoff at the Academic Medical Center, Amsterdam ** Kindly provided by A. te Velde at the Academic Medical Center, Amsterdam
mRNA Primer Sequences (5′-3′)
18S rRNA Forward primer AAT CTG GAG CTG GCC TTT CA Reverse primer CTG GAA GAT CTG CAG CCT TT
IL5 Forward primer AGC TGC CTA CGT GTA TGC CA Reverse primer CAG GAA CAG GAA TCC TCA GA
IL13 Forward primer ATT GCT CTC ACT TGC CTT GG Reverse primer GTC AGG TTG ATG CTC CAT ACC
TNF* Forward primer TGC TTG TTC CTC AGC CTC TT Reverse primer TGG GCT ACA GGC TTG TCA CT
IL17RA** Forward primer ATC CTG CTG GTG GGC TCC GT Reverse primer ACG TAG AGG GGG TGG TCG GC
IL17RB Forward primer CCA ACA CAG CAC TAT CAT CG Reverse primer ATA TGG AGT CAG CTG CAC CG
IL1RL1 (ST2) Forward primer ATG TTC TGG ATT GAG GCC AC
Reverse primer GAC TAC ATC TTC TCC AGG TAG CAT
IL23R Forward primer AAC AAC AGC TCG GCT TTG GT Reverse primer GGA ATA TCT GGC GGA TAT CC
AHR Forward primer CTT AGG CTC AGC GTC AGT TA Reverse primer GTA AGT TCA GGC CTT CTC TG
METHODS
Fetal and adult tissues
Human fetal tissues were obtained from elective abortions at the Stichting Bloemenhove clinic in Heemstede, the Netherlands, upon on the receipt of informed consents. The use of human abortion tissues was approved by the Medical Ethical Commission of the Academic Medical Center, Amsterdam. Gestational age was determined by ultrasonic measurement of the diameter of the skull or femur and ranged from 14-17 weeks.
Adult non-inflamed nose conchae tissue was obtained from healthy individuals. Inflamed nose polyps were from chronic rhinosinusitis patients. Adult lung tissues were obtained after informed consents from patients undergoing lung tumor surgery, where the tissues were obtained at a clear distance from the tumor. Collection of lung and nose tissue was approved by the Medical Ethical Commission of the Academic Medical Center, Amsterdam. Inflamed ileum tissue was collected from Crohn’s disease patients undergoing resection surgery. Non-inflamed ileum was obtained from patients undergoing colon tumor resection surgery from which ileum tissue was collected at a clear distance from the tumor. Both inflamed and non-inflamed ileum tissue was obtained as residual material after clinical procedures according to the ethical guidelines at the Academic Medical Center (AMC), Amsterdam, the Netherlands.
Isolation of cells
All solid tissues were rinsed of connective tissue, fat and muscle. Intestinal tissues were also cleared from meconium after which the adult ileum tissue was incubated with dithiothreitol (154 µg/ml), 0.1% β-mercaptoethanol and 5 mM EDTA for elimination of epithelial cells and mucus. The tissues were cut into fine pieces and digested for 30-45 min at 37 °C with Liberase TM (125 µg/ml) and DNase I (200 µg/ml) (both from Roche). Lung cells were isolated by incubating the tissue with 50 U/ml DNAse type I (Sigma-Aldrich) and collagenase type I 300 U/ml (Worthington). The cell suspensions were filtered through a 70-µm nylon mesh or equivalent and mononuclear cells were isolated using Ficoll Paque Plus (GE Healthcare). PBMC were isolated on Lymphoprep (Nycomed) or Ficoll-Paque.
Flow cytometric analysis and sorting
The following anti-human antibodies were used (clone name within brackets):
FITC-conjugated anti-CD4 (RPA-T4), CD14 (MφP9), CD16 (3G8), CD19 (HIB19), CD34 (581), CD56 (NCAM16.2), TCRαβ (IP26), TCRγδ (B1), PE-conjugated anti-CD16 (3G8), CCR4 (1G1), CXCR3 (IC6/CXCR3), AF647-conjugated anti-CRTH2 (CD294; BM16), CXCR5 (RF8B2), APC-cyanine 7 (Cy7) anti-CD45 (2D1) and isotypes Alexa Fluor 700 (MOPC-21), PE, APC, PECy7 (X40) (all from Beckton Dickinson), APC-conjugated anti-CD4 (S3.5) (Invitrogen), APC-conjugated anti-CD45RA (ALB11), PE-cyanine 7 (Cy7) human CD127 (R34.34) (Beckman Coulter), PE-conjugated anti-RORγt (AFKJS-9) (eBioscience), PE-conjugated anti-IL-1R1 (goat polyclonal) (R&D), FITC-conjugated anti-human BDCA2 (CD303; AC144; Milenyi).
For flow cytometric phenotype analysis, data were acquired on an LSRFortessa or LSRII (BD) and analyzed with FlowJo software (TreeStar, Inc.).
For flow cytometry sorting, PBMC were depleted of T, B, NK cells and monocytes by labeling with FITC-conjugated anti-CD3, CD14, CD16 and CD19 antibodies (described above) plus anti-FITC microbeads (Miltenyi) or the corresponding EasySep antibodies plus beads (StemCell Technologies). Lin–CD127+CRTH2+ ILCs from fetal gut and peripheral blood were sorted on a FACSAria (BD) to ≥ 98% purity.
Establishment of CD127+CRTH2+ cell lines and analysis of cytokine production
ng/ml; R&D Systems), IL-33 (50 ng/ml; R&D Systems) and combinations of these cytokines. Fresh cells were stimulated with PMA plus ionomycin for 24 h. Expanded cells were stimulated with IL-2, IL-1β (Miltenyi), IL-23 (R&D) or combinations of these cytokines for 3-4 days. Supernatants were analyzed for presence of IL-13 and IL-22 with ELISA (Sanquin and Genentech-Roche in-house or R&D, respectively). Multiple cytokine detection was performed in some experiments using Luminex (Biorad).
Intracellular cytokine staining
Ex vivo expanded cell lines were stimulated for 6 h with 10 ng/ml PMA (Sigma) and 500 nM ionomycin (Merck) in the presence of Golgiplug (BD) or 5 g/ml BFA for the final 4 hours of culture. Cell permeabilization, staining, and subsequent washings were performed using the Cytofix/cytoperm kit (BD). The following antibodies were used: APC-conjugated anti-IL-13 (JES10-5A2, BioLegend), APC-conjugated IL-17 (BL168, BioLegend) and PE or Alexa647–conjugated anti-IL-22 (142928, R&D or 3F11, Genentech-Roche36, respectively), anti-IFN-γ (B27. BD Bioscience) or anti-TNF (MAb11, BD Bioscience). Data were acquired on an LSRFortessa or LSRII (BD) and analyzed with FlowJo software (Tree Star, Inc.).
Quantitative real-time PCR
Instrument II (Roche). In house designed primers are given in Supplementary Table 1. Primers for IL17, IL22 and IFNG were used as previously published4. The LinRegPCR software37,38 was used for quantification of expression. All samples were normalized using 18S rRNA expression and expressed in arbitrary units.
Statistical analysis
Differences in nasal tissue CRTH2+ ILC frequencies between healthy controls and chronic rhinosinusitis patients were calculated using two-tailed Mann-Whitney U-test.
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